Biochemical species targeted in therapy or diagnosis frequently include cell surface antigens, which, upon recognition by natural or synthetic binding molecules, trigger a network of signal transduction and gene regulation events inside the cell that result in cellular responses important in the initiation or maintenance of a disease. Target antigens may also differentially reside on the surface of different cells and signify a unique state of physiology or disease progress in the tissue or organ.
Monoclonal antibodies directed against tumor-associated antigens expressed on the tumor cell surface have found application in the immunotherapy of human tumors. The interaction of certain monoclonal antibodies directed against tumor cells with cell surface antigens is well documented. See, e.g., Herlyn et al. (1987), Koprowski et al. (1985), Grob et al. (1985), and Murthy et al. (1987). Monoclonal antibodies targeted to the epidermal growth factor receptor (EGFR) are of great interest, due to the high level of expression of this receptor in patients with solid tumors. MAB have also been developed which target molecules implicated in other diseases, such as autoimmune diseases, AIDS, asthma, psoraisis, and other inflammatory disorders.
Target-specific binders such as antibodies are also widely employed in detection of such biological markers, particularly in differential assays of markers in different cell or tissue types, e.g. in immunohistochemical assays. Immunohistochemistry (IHC), which broadly includes techniques of screening for target analytes in multiple tissue or cell samples, such as tissue libraries, by application of known binding molecules followed by detection, can be used for detection of the presence or absence of molecular markers, typically proteins, involved in various stages of diseases, such as cancer. Methods have been developed for carrying out such assays on surface antigens in whole cells, as opposed to soluble antigens (see e.g. Bishop & Hwang, 1992; Bator & Reading, 1989). Over the last few years, the role of immunohistochemical analysis has been changing from that of an ancillary diagnostic technique to that of a stand-alone diagnostic method (O'Leary 2001). IHC studies play an increasingly important role in surgical pathology, and can be used, for example, in classifying tumors, predicting the origin of a carcinoma, demonstrating micrometastases and microorganisms, providing prognostic information, or rendering a diagnosis in a damaged specimen (Chan 2000).
In standard IHC assays, markers are generally detected by immunostaining of the specific binding molecules (e.g. antibodies) added to the sample. Consistency of interpretation of staining results, especially from one clinical setting to another, can be problematic. This technique also limits the number of different markers which can be detected simultaneously.
Many receptors are normally expressed on the cell surface to only a small extent, i.e. a few hundred to a few thousand receptors/cell. These include many receptors types of great medical interest, including, for example, receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), many interleukins, erythropoietin, and tumor necrosis factor (TNF). In addition, assays must often be carried out on tissue samples which are small, rare, unique, or otherwise limited in quantity. Accordingly, there is a need for methods of detecting such markers, often present at low levels, in limited tissue samples, where the method is quantitative, reproducible, and high sensitivity; that is, it gives a readily detectable signal from a small amount of analyte.